The term microvesicles (also known as microparticles) refers to a heterogeneous in vivo collection of membrane bound (i.e., encapsulated) biological structures. These structures are formed from lipid bilayer, which is the same lipid bilayer that comprises eukaryotic cell membranes. Microvesicles can reside within the cell, or in the extracellular environment. Microvesicle structures (intracellular and/or extracellular) are produced by nearly all mammalian cell types, as well as during in vitro cell culture.
The molecular composition of microvesicles is diverse, containing and/or transporting a variety of nucleic acids, proteins and lipids. Microvesicle molecular composition is generally reflective of the plasma membrane and antigenic content of the cell types, tissues and organs from which they originate. Mathivanan and Simpson, “Exosomes: extracellular organelles important in intercellular communication,” J. Proteomics 73(10):1907-1920 (2010). Although protein composition of the microvesicles varies, most of these structures are enriched for various soluble protein markers, including HSP70, Hsc70, CD63, CD9, CD81 and others. Circulating microvesicles have also been reported to contain nucleic acids, including messenger RNAs, and relatively high levels of small RNAs and microRNAs.
Circulating microvesicles are associated with numerous cell functions, including intercellular (cell-to-cell) communication, removal of metabolic byproducts and toxins (including misfolded proteins, cytotoxic agents and metabolic waste), angiogenesis, tissue regeneration, endocytic recycling of the plasma membrane, selective removal of plasma membrane proteins and regulation of immune functions such as antigen presentation. Some microvesicles have been shown to transport messenger RNA (mRNA) and microRNA (miRNA), which is highly suggestive of microvesicles functioning as messengers that allow one cell type to regulate the activity of a distant cell type by acting as a shuttle that can merge with the distant cell and release its contents into that target recipient cell. This microvesicle shuttle can utilize the body fluids to travel to distant sites and control the activity of distant target cells.
Circulating microvesicles (cMVs), or synonymously, extracellular microvesicles (eMVs), describe an eclectic group of microvesicles that are released by cells, and therefore, exist in extracellular spaces and/or reside in body fluids. The mammalian body fluids that are known or suspected to contain cMVs include, but are not limited to, blood, urine, ascites fluid and cerebrospinal fluid. Secreted microvesicles are also found in cell culture media that has been exposed to cultured mammalian cells.
With regard to defining and categorizing the cMV molecules that can be found in body fluids, there is lack of consensus as to the nomenclature and description of the different types of cMV particles. Some literature distinguishes at least three subcategories of circulating microvesicles, based on their mechanistic origin. The molecular/cellular mechanisms that produce microvesicles are theorized to include (i) exocytosis of intracellular multivesicular bodies, (ii) outward budding, fission and shedding of plasma membrane, and (iii) byproducts of apoptosis. The diverse collection of circulating microvesicle structures can range in size from about 20 nanometers (nm) to upwards of about 1,000 nm (i.e., 1.0 micrometer, micron, or μm) in diameter.
The first recognized subgroup of cMVs are those produced by direct plasma membrane budding, fission and shedding. Some sources describe these shed microvesicles as generally large, namely with lower sizes limits of at least 100 nm or 200 nm, and with an upper size limit of about 1,000 nm in diameter. Some have proposed that these structures be termed “ectosomes” or “shedding microvesicles (SMVs).” Still other groups state that ectosome particles may be as small as 40 or 50 nm in diameter.
A second recognized subgroup of cMVs are exosomes, that is, the preformed microvesicles that are released from the cell following the exocytic fusion of intracellular multivesicular bodies with the plasma membrane. These exosome structures are generally smaller than ectosmoes, and have an upper size limit estimated to be about 100, 150 or 200 nm, and a lower size limit of about 40 nm or 50 nm. However, various sources differ in their size-based definitions for exosomes, and this size distinction remains unresolved.
A third group of structures is the apoptotic blebs released by dying cells. These membrane structures have a less well defined size range, and may be anywhere from about 50 nm to about 5,000 nm in diameter.
A unified microvesicle nomenclature and classification system utilizing broadly accepted definitions has been elusive in the field. In the literature, microvesicles have been alternatively referred to as microparticles, nanoparticles, exosomes, ectosomes, epididimosomes, argosomes, exosome-like vesicles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes, oncosomes, exosome-like vesicles, apoptotic blebs, and shedding microvesicles. In some publications, uses of these terms is conflicting or overlapping. Simpson and Mathivanan (2012), “Extracellular Microvesicles: The Need for Internationally Recognized Nomenclature and Stringent Purification Criteria”. J Proteomics Bioinform (2). doi:10.4172/jpb.10000e10. One source suggests that a preferred nomenclature for circulating microvesicle is based on the microvesicle's mechanism of origin. Namely, these categories would be (i) the ectosomes produced by membrane budding, (ii) the exosomes produced by the exocytosis to intracellular multivesicular bodies, and (iii) the membrane blebs produced by the process of apoptosis.
The function of extracellular microvesicles is not clearly understood, although they are theorized to act as nano-shuttles for the transport and delivery of information from one location and/or cell type to distant locations and/or other cell types. Exosomes are theorized to be involved in a wide variety of physiological processes, including cardiac disease, adaptive immune responses to pathogens, and in tumor biology. It is suggested that microvesicles may play roles in tumor immune suppression, metastasis, and tumor-stroma interactions. Microvesicles are thought to play a role in immune system cellular communication, for example, involving dendritic cells and B cells.
The ubiquitous presence of circulating microvesicles in body fluids, their association with a broad range of physiological processes, as well as their elevated levels in human disease, suggest that microvesicles can potentially serve as tools in molecular medicine as measures of physiological state, disease diagnostics, and possibly therapeutic targeting.
Although the study of microvesicles/exosomes had been greatly advanced with the development of analytical systems such as nanoparticle tracking analysis (NTA) and fluorescent nanoparticle tracking analysis (FNTA), other technical challenges remain.
One of the significant technical challenges in current research in microvesicles is the problem of how to efficiently isolate the microvesicles from various sources. Current methodologies to isolate secreted microvesicles, including but not limited to exosomes, are constrained by technical limitations and other drawbacks. These known methodologies are labor intensive, time-consuming and costly. See (i) Van der Pol et al., “Optical and non-optical methods for detection and characterization of microparticles and exosomes,” Journal of Thrombosis and Haemostasis (2010), doi: 10.1111/j.1538-7836.2010.04074.x; (ii) Dragovic et al., “Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis,” Nanomedicine: Nanotechnology, Biology and Medicine (2011), doi:10.1016/j.nano.2011.04.003; and (iii) Tauro et al., “Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes,” Methods 56(2):293-304 (print February 2012, Epub Jan. 21, 2012), doi: 10.1016/j.ymeth.2012.01.002.
Historically, ultracentrifugation is the traditional method for microvesicle isolation. Generally, centrifugation is the process whereby a centrifugal force is applied to a mixture, whereby more-dense components of the mixture migrate away from the axis of the centrifuge relative to other less-dense components in the mixture. The force that is applied to the mixture is a function of the speed of the centrifuge rotor, and the radius of the spin. In most applications, the force of the spin will result in a precipitate (a pellet) to gather at the bottom of the centrifuge tube, where the remaining solution is properly called a “supernate” or “supernatant.” In other similar applications, a density-based separation or “gradient centrifugation” technique is used to isolate a particular species from a mixture that contains components that are both more dense and less dense than the desired component (e.g., OptiPrep™).
During the circular motion of a centrifuge rotor, the force that is applied is the product of the radius and the angular velocity of the spin, where the force is traditionally expressed as an acceleration relative to “g,” the standard acceleration due to gravity at the Earth's surface. The centrifugal force that is applied is termed the “relative centrifugal force” (RCF), and is expressed in multiples of “g” (or “×g”).
The centrifugation procedures that have been used to isolate circulating microvesicles can incorporate as many as five centrifugation steps, with at least two of these spins requiring centrifugal forces in excess of 100,000×g for several hours. Generally, ultracentrifugation is centrifugation conditions that produce forces in excess of 100,000×g. These ultracentrifugation procedures are time consuming and labor intensive, and furthermore, are constrained by the requirement for expensive ultracentrifugation equipment.
Size exclusion chromatography can also be used to isolate microvesicles, for example, by using a Sephadex™ G200 column matrix. This approach is also time consuming and the yields are inconsistent.
Selective immunoaffinity capture (including immuno-precipitation) can also be used to isolate circulating microvesicles, for example, by using antibodies directed against the epithelial cell adhesion molecule, a type-I transmembrane cell-surface protein (also known as EpCAM, CD326, KSA, TROP1). The anti-EpCAM antibodies can be coupled to magnetic microbeads, such as Dynabeads® magnetic beads. This method has very low yields compared to other methods, and is costly due to the use of the immuno-reagents and magnetic beads, and further, these system reagents cannot be reused for subsequent isolations.
The in vitro culture of mammalian cells is a critical tool in the study of cell biology, including microvesicle function. Historically, mammalian cells (including both adherent and non-adherent lines) are cultured using a defined minimal media supplemented with a blood serum or blood plasma. There exists a wide variety of minimal growth media, which a user would select to optimize the growth of the particular cell line of interest. Examples of classic defined media include Basal Medium Eagle (BME), Dulbecco's Modified Eagle's medium (DMEM), minimal essential medium (MEM), F10 Nutrient Mixture, Ham's tissue culture medium, Ham's F12 medium and RPMI-1640 medium. Countless variations of minimal defined media have been developed since the original formulations were developed. In addition, individual laboratories will often develop their own unique formulations in order to optimize their particular experimental system.
These minimal media contain various concentrations of defined components, including, for example, but not limited to, the 20 amino acids, purine and pyrimidine nucleotides and/or nucleotide precursors, phospholipids and phospholipid precursors, vitamins (as parts of coenzymes), lipoic acid, a carbon source such as glucose, and inorganic ions. Some formulations add additional components, such as growth factors and hormones, and/or vary the concentrations of the various components.
Complete growth medium for most in vitro cultured mammalian cells requires supplementation of the minimal defined media with a blood serum or blood plasma, most typically heat inactivated blood sera. This supplementation is typically on the order of 1% to 20% blood serum by volume of the minimal defined media.
The serum that is used to supplement the minimal defined medium can be from a variety of sources, for example, bovine, equine (horse), human, mouse, rat and goat. Bovine serum is most commonly used in laboratory settings. Serum supplements that are derived from age-staged animals can also be used, and may be desirable for their various growth properties. For example, bovine serum can be age-staged as fetal bovine serum (FBS), calf serum (CS), newborn calf serum, or adult bovine serum. Heat inactivated FBS is frequently used in many applications. Heat inactivated FBS is frequently used in combination with DMEM to form a complete growth media for many types of mammalian cells.
It is known that FBS contains an abundance of endogenous bovine microvesicles, including exosomes. These endogenous microvesicles can exert effects on cultured cells when the FBS is used as a supplement to make a complete culture medium. Sakwe et al., “Fetuin-A (alpha-2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells,” J Biol. Chem. 285(53):41827-41835 (2010). These endogenous microvesicles may impact the growth or differentiation of cells maintained in culture, and may skew or interfere with experimental results and experimental interpretation. In other examples, the endogenous microvesicles found in blood serum can copurify with microvesicles that are produced by the cultured cell lines of interest. Bhatnagar et al., “Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo,” Blood Vol. 110, no. 9:3234-3244 (2007).
Although some cultured cell lines can be grown for a short time in minimal media in the absence of a serum supplement, this is not always possible. For these reasons, it would be desirable to remove or deplete cell culture media of any endogenous microvesicles that are introduced through the traditional addition of blood serum or blood plasma components in cell culture media.
What is needed in the art are methods for the rapid and inexpensive isolation of microvesicles, specifically circulating microvesicles, for example, exosomes and microsomes. Preferably, these methods will utilize common laboratory reagents and apparatus, and will not require high speed centrifugation, such as ultracentrifugation. What is needed in the art are methods for the isolation of circulating microvesicles, where the methods utilize low speed centrifugation that uses centrifugal forces significantly less than 100,000×g.
Furthermore, what is also needed in the art are methods for generating cell culture media that are free of endogenous microvesicles, or have reduced concentrations of endogenous microvesicles compared to traditional complete media. What is needed in the art are methods for generating blood serum or blood plasma media supplements that are free of endogenous microvesicles, or have reduced concentrations of endogenous microvesicles compared to untreated blood serum or plasma.